Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammalian brain and has been studied in connection with a number of nervous, mental and autoimmune disorders. The long-term goal of these investigations is to determine how GABA synthesis is regulated by glutamate decarboxylase )GAD). Rat brain GAD exists as two proteins, GAD67 and GAD65, with molecular weights of 67 and 65 kDa respectively, that are the products of two separate genes. Emerging evidence suggests that the expression of GAD67 and GAD65 is differentially regulated; GAD65 through its interaction with the co-factor pyridoxal phosphate and GAD67 at the level of protein expression. The studies proposed here focus on the regulation of the expression of GAD67. Total GAD activity and levels of GAD67 protein in adult rat cerebral cortex and in cultured, GABAergic neurons are decreased by treatment with the GABA-transaminase inhibitor gamma-vinyl-GABA (GVG). The levels of GAD65 protein remain unchanged. GAD67 mRNA levels are not changed by GVG treatment, suggesting a diminished translation of GAD67 mRNA and/or a change in the turnover of GAD67 protein. Results of preliminary experiments indicate that GVG induces a dissociation of GAD67 mRNA from the heavy polysomes. I intend to address the biological relevance of GABA's action on GAD67 by determining the sensitivity of GAD67 to intracellular GABA levels (Aim 1). I will also further examine the mechanism of GABA's posttranscriptional regulation of GAD67 (Aims 2 and 3). This will be accomplished by analyzing the distribution of GAD67 mRNA on polysomes and by determining the half-lives of the GAD proteins with 35S-methionine pulse-labeling experiments. In addition to these experiments I will investigate the actions of estradiol on GAD67 expression (Aims 4 a-e). I have determined that estradiol decreases total GAD activity in cultured GABAergic neurons and I will investigate whether estradiol influences GAD protein and mRNA levels. Subsequently, I will determine if estradiol influences GAD mRNA translation, protein stability, mRNA stability and/or GAD gene transcription. I will use nuclear run-off assays to determine the transcriptional activity and perform experiments with transcription inhibitors to determine the stabilities of the GAD mRNA's. It has been reported that estradiol decrease GAD67 mRNA levels in the brain and it is well-known that estradiol act at the level of nuclear DNA to regulate gene expression.